TERT promoter mutations in urothelial neoplasia

ABSTRACT

TERT promoter mutations occur in both papillary and flat lesion bladder cancers, are the most frequent genetic alterations identified to date in noninvasive precursor lesions of the bladder, are detectable in urine, and appear to be strongly associated with bladder cancer recurrence. The TERT promoter mutations are useful urinary biomarker for both the early detection and monitoring of bladder neoplasia.

This invention was made with funding from the U.S. national institutesof Health, grant numbers CA43460 and CA57345. The government thereforeretains certain rights in the invention.

The disclosures of prior applications U.S. Ser. No. 61/881,113 filed 23Sep. 2013, PCT/US14/51808 filed 20 Aug. 2014, U.S. Ser. No. 61/765,909filed 18 Feb. 2013, U.S. Ser. No. 61/766,857 filed 20 Feb. 2013, U.S.Ser. No. 61/772,249 filed 4 Mar. 2013, PCT/US14/16906 filed 18 Feb.2014, and U.S. Ser. No. 14/765,692 filed 4 Aug. 2015 are expresslyincorporated herein.

TECHNICAL FIELD OF THE INVENTION

This invention is related to the area of cancer. In particular, itrelates to urothelial cancers.

BACKGROUND OF THE INVENTION

Urothelial carcinoma of the bladder is the most common malignancy of theurinary tract with 73,000 new cases and 15,000 deaths expected in 2013in the US alone (1). These invasive carcinomas arise from histologicallywell-defined papillary and flat precursor lesions, providing a potentialopportunity for early detection and treatment (2). Although urinecytology enjoys a reasonable sensitivity and specificity for detectinghigh-grade neoplasms, its performance in detecting low-grade tumors ispoor, with a sensitivity and specificity of 4% and 30%, respectively(3).

A number of urine-based markers have been developed to improve theaccuracy of noninvasive screening and surveillance in bladder cancer.Among Food and Drug Administration (FDA) approved tests, the Immunocytest (Scimedx Corp, Danville, N.J.), nuclear matrix protein 22 (NMP22)immunoassay test (Matritech, Cambridge, Mass.) and multitargetfluorescence in situ hybridization (FISH) (UroVysion; Abbott Park, Ill.)(4) have demonstrated an overall sensitivity of 70% and a specificityrange of up to 89%. Performance inconsistencies, as a result ofvariability in pre-analytical and analytical specimen factors, haveimpeded their wide-spread clinical use.

Activating mutations in the promoter of the telomerase reversetranscriptase (TERT) gene lead to increased telomerase expression and,in doing so, allow some neoplasms to overcome the end-replicationproblem and avoid senescence. TERT promoter mutations were initiallydescribed in melanoma (5, 6) and have subsequently been described in adiscrete spectrum of cancer types, including 66% of muscle-invasiveurothelial carcinomas of the bladder (5, 7). TERT is therefore the mostfrequently mutated gene in advanced forms of this disease, and thelocalization of these mutations to a small gene region in the TERTpromoter provides an extraordinary opportunity for biomarker development(7).

Muscle-invasive urothelial carcinoma is responsible for the vastmajority of bladder cancer related deaths and many of these deaths couldbe prevented if precursor lesions were detected and surgically excisedprior to their invasion into the muscle (10-13). New strategies for theearly detection of such lesions are therefore urgently needed (14).There is a continuing need in the art to find ways of detecting early,curable, bladder disease and for monitoring recurrences of bladdercancer after tumor resection.

SUMMARY OF THE INVENTION

According to one embodiment of the invention a method is provided.Nucleic acids obtained from a urine sample of a human are tested for agenetic alteration in telomerase reverse transcriptase promoter at oneor more nucleotides between 1295205 and 1295297 on the minus strand ofchromosome 5 in version hg19 of human genome sequence. The human has notbeen diagnosed with bladder cancer.

According to another aspect of the invention a method is provided.Nucleic acids obtained from a urine sample of a patient are tested for agenetic modification in telomerase reverse transcriptase promoter. Oneor more nucleotides between 1295205 and 1295297 on the minus strand ofchromosome 5 in version hg19 of human genome sequence are interrogated.The patient has had a surgical excision or other treatment for a bladdercancer.

Another aspect of the invention is two primer pairs. In the first pair,a first primer comprises a first segment of TERT promoter regiongcggaaaggaaggggag (SEQ ID NO: 5), and a second primer comprises a secondsegment of TERT promoter region CCGTCCCGACCCCT (SEQ ID NO: 6). In thesecond pair, a first primer comprises a first segment of TERT promoterregion ggccgcggaaaggaag (SEQ ID NO: 7), and a second primer comprises asecond segment of TERT promoter region CGTCCTGCCCCTTCACC (SEQ ID NO: 8).

These and other embodiments which will be apparent to those of skill inthe art upon reading the specification provide the art with

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic of the TERT locus and positioning of theSafe-SeqS amplification primers. The yellow marks indicate the positions(offset by −1,295,000 base pairs) of the most common TERT promotermutations previously reported and identified in this study. UID: Uniqueidentifier; UPS: Universal primer binding site.

FIG. 2=Table 1: Clinicopathologic characteristics of patients analyzedin this study

FIG. 3=Table 2: TERT promoter mutations

FIG. 4=Table 3: Correlation between TERT promoter mutation status andtumor recurrence.

FIG. 5=Table 4: Correlation of TERT promoter mutation status and tumorprogression

FIG. 6=Table 5: Correlation of TERT mutation status in originaldiagnostic transurethral resection biopsy (TURB) tissue and TERTmutation status in urine collected at follow-up.

FIG. 7=Table 6: Correlation of TERT promoter mutation status infollow-up urine samples with recurrence

FIG. 8=Table S1. TERT promoter mutation status in 59 pTa and 17carcinoma in situ (CIS) patients.

DETAILED DESCRIPTION OF THE INVENTION

The inventors have developed a method for detecting TERT promotermutations in urine.

The presence of these mutations in urine is strongly associated withbladder cancer recurrence in patients who have had their tumors removedsurgically. Moreover, TERT promoter mutations are the most commongenetic alteration in noninvasive bladder cancer identified to date,occurring in the majority (74%) of such precursor lesions. They occur incancers developing through both the papillary and flat routes to tumorprogression (15), and occur in low-grade as well as high-grade tumors.These mutations can be detected in the urine of patients with bladdercancer using sensitive techniques we have developed. TERT promotermutations provide a useful biomarker for the early detection of bladdercancers, and patients at high risk for this disease can be screened inthis noninvasive way.

Given the high prevalence of TERT promoter mutations in early bladderneoplasia, their presence or absence in tumors is of limited prognosticvalue. However, superficial bladder cancers are currently the mostcostly solid tumor (per patient) in the U.S. (16, 17). Noninvasivemethods to monitor these patients could reduce the cost of caring forthese patients as well as the discomfort associated with invasiveprocedures. Among patients with TERT mutations in their primary tumors,there was a highly significant correlation between the presence ofmutations in subsequent urine collections and recurrence (Table 6).

Our results therefore indicate two avenues for application of TERTpromoter mutations in the clinic: early detection in high-risk patientsand monitoring of patients with bladder cancer, both through theanalysis of urine specimens. TERT promoter mutations occur early, arespecific for neoplasia, and can be identified in the urine withtechniques such as those described below.

Testing nucleic acids can be accomplished by any means known in the artfor determining a nucleotide identity at one or more positions. Testingtypically involves isolation of nucleic acids from a clinical sample anddoing at least one transformative reaction on the nucleic acids.Alternatively, a sample can be treated to make its nucleic acidsaccessible to probes, to perform an in situ assay. For example, thenucleic acids can be denatured and hybridized to a probe. The nucleicacids can be amplified. The nucleic acids can be hybridized to a primerand the primer extended by one or more bases. The nucleic acids can bemodified by an enzyme that uses DNA as a substrate. Each of thesemethods involves a transformation of nucleic acids. The nucleic acidsare a physical substance and are not a representation. One or bothstrands can be tested or assayed.

Therapies which can be prescribed are any that are known in the art fortreating bladder cancer. These may include Bacillus Calmette-Guérin(BCG) and intravesical chemotherapy. Adjuvant therapies may includechemotherapy agents such as cisplatin and gemcitabine. Otherchemotherapy agents or biological agents can be used in combination orseparately. Similarly, radiation can be used alone or together withother therapies. Prescribing a therapy typically involves a medicalprofessional making a determination based on fact or surmise that aparticular therapy will be or might be efficacious. The medicalprofessional typically permanently records this determination in amedical chart or file. In addition, the drug and dosage are reduced to averbal communication, such as a writing, a voice recording, or anelectronic message that is delivered to a dispensing pharmacy.

Any confirmatory test can be performed as is known in the art if abladder cancer is detected by means of the urine test. One such test isa cytoscopy. Any test that can sensitively detect a bladder cancer canbe used.

Elevated risk of a bladder cancer can be ascertained based on knownexposure to a carcinogen. Such exposures may be, for example,environmental, nutritional, or pharmaceutical. Cigarette or othertobacco smoking or ingesting, which may be considered an environmentalexposure, is a major risk factor. Alternatively or in addition, theelevated risk may be due to a family history of bladder cancer.

Primers according to the invention are complementary to portions of theTERT promoter region. Preferably the primer will comprises a sequenceaccording to any of SEQ ID NOs: 5, 6, 7, or 8. Primers will typically beat least 14, 15, 16, 17, 18, 19, 20, or 21 nucleotides in length andtypically will be less than 50, 45, 40, 35, 30, 25 nucleotides inlength. The primers will also comprise sequences that are notcomplementary to portions of the TERT promoter. Thus any of thespecified TERT promoter sequences will be linked to other non-TERTpromoter sequences. For example, the TERT promoter sequences may belinked to a universal priming site (UPS) and/or a unique identifier(UID). The UID may, for example be comprised of a number of degenerate Nbases (equal likelihood of being an A, C, T, or G). The degenerate basesmay range from 4-20, typically. Thus the sequence of the primers is notnaturally occurring. In some circumstances, the primer may be linked toa non-nucleotide moiety, such as to a fluor, a chromophor, or aradioactive moiety.

The above disclosure generally describes the present invention. Allreferences disclosed herein are expressly incorporated by reference. Amore complete understanding can be obtained by reference to thefollowing specific examples which are provided herein for purposes ofillustration only, and are not intended to limit the scope of theinvention.

Example 1 Materials and Methods

Patient Samples

This study was approved by the Institutional Review Board of JohnsHopkins University, School of Medicine. Two different sets of sampleswere analyzed in our study. The first sample set included 76 noninvasivepapillary urothelial carcinomas and flat carcinoma in situ (CIS) lesionsobtained by transurethral bladder resection (TURB) between 2000 and2012. All specimens were rom the Surgical Pathology archives and wereselected only on the basis of specimen availability. Pertinent patientdemographics and clinical information were obtained from electronicmedical records. All sections were reviewed by three urologicalpathologists (EM, SFF and GJN) to confirm the original diagnoses. Toenrich for neoplastic cells within the tissues, representativeformalin-fixed paraffin-embedded (FFPE) blocks were cored with a sterile16 gauge needle and tumor areas showing at least 50% neoplasticcellularity were selected microscopically. For eight of the cases,benign adjacent urothelium was macrodissected from FFPE blocks. Thecores were placed in a 1.5 mL sterile tube for subsequent DNApurification using an AllPrep DNA/RNA Mini Kit (Qiagen, cat. no. 80204).DNA was purified from peripheral blood buffy coats of 15 patients usingthe same Qiagen kit.

For the second sample set, we prospectively collected urine samples from15 separate patients undergoing follow-up cystoscopy for previouslydiagnosed non-muscle-invasive urothelial carcinoma. We purposely biasedthis cohort to include patients that recurred within the follow-upperiod. Immediately prior to follow-up cystoscopy, 25 mL of raw urinewas collected and subsequently pelleted by centrifugation at 3,000 g for10 minutes. The pellets were stored at −80° C. in 1.5 mL tubes forsubsequent DNA extraction. For 14 of these patients, matched FFPE fromthe original diagnostic TURB was retrieved. These included 13 high-gradeurothelial carcinomas (pTa HG and pT1 HG in six and seven cases,respectively), and one low-grade papillary urothelial carcinoma (pTaLG). Twenty 8 μm-thick sections were cut from one representative tissueblock in each case and areas containing at least 70% neoplastic cellswere microdissected and used for DNA purification using a QIAamp DNAFFPE Tissue Kit (Qiagen, cat no. 56404).

Mutation Analysis

Due to their tremendous throughput, massively parallel sequencinginstruments are highly cost-effective for DNA mutation analysis.However, sample preparation and sequencing steps introduce artifactualmutations into analyses at a low, but significant frequency. To betterdiscriminate genuine TERT promoter mutations from artifactual sequencingvariants introduced during the sequencing process, we used Safe-SeqS, asequencing error-reduction technology described previously (8, 9). Asdepicted in FIG. 1, Safe-SeqS amplification primers were designed toamplify a 126-bp segment containing the region of the TERT promoterpreviously shown to harbor mutations in melanomas and other tumors(5-7). The forward and reverse amplification primers contained theTERT-specific sequences at their 3′ ends and a universal priming site(UPS) at their 5′ end. The reverse primer additionally contained a14-base unique identifier (UID) comprised of 14 degenerate N bases(equal likelihood of being an A, C, T, or G) between the UPS andgene-specific sequences. The sequences of the forward and reverseprimers were either

(SEQ ID NO: 1) 5′-CACACAGGAAACAGCTATGACCATGGGCCGCGGAAAGGAAG and(SEQ ID NO: 2) 5′-CGACGTAAAACGACGGCCAGTNNNNNNNNNNNNNNCGTCCT GCCCCTTCACC,or (SEQ ID NO: 3) CACACAGGAAACAGCTATGACCATGGCGGAAAGGAAAGGGAG and(UPS sequences underlined; SEQ ID NO: 4)5′-CGACGTAAAACGACGGCCAGTNNNNNNNNNNNNNNCCGTCC CGACCCCT.

These primers were used to amplify DNA in 25 μL PCR reactions in 1×Phusion Flash High-Fidelity PCR Master Mix (Thermo Scientific, cat. no.F-548L) containing 0.5 μM forward and reverse primers (described above).After incubation at 98° C. for 120 seconds, 10 cycles of PCR wereperformed in the following manner: 98° C. for 10 seconds, 63° C. for 120seconds, and 72° C. for 120 seconds was performed. Reactions werepurified with AMPure XP beads (Beckman Coulter) and eluted in 100 μL ofBuffer EB (Qiagen, cat. no. 19086). For the second stage ofamplification, 5 μL of purified PCR products were amplified in 25 μLreactions containing 1× Phusion Flash High-Fidelity PCR Master Mix and0.5 μM amplification primers that each contained the first-stage UPS attheir 3′ ends and the grafting sequences required to hybridize to thesequencing instrument flow cell at their 5′ ends (8, 9). The reverseamplification primer additionally included a 6 bp index sequence, uniqueto each sample, inserted between the UPS and grafting sequences. Afterincubation at 98° C. for 120 seconds, 17 cycles of PCR were performed inthe following manner: 98° C. for 10 seconds, 63° C. for 120 seconds, and72° C. for 120 seconds. The PCR products were purified with AMPure andsequenced on a MiSeq instrument.

Data were analyzed as previously described (8, 9). Briefly, theamplified TERT promoter region of reads containing UIDs, where each baseof the UID region had instrument-derived quality scores ≥15, was matchedto a reference sequence using a custom script. TERT promoter sequenceswith five or fewer mismatches were retained for further analysis. Tumorsamples were considered positive if the fraction of mutations exceeded1% of alleles (which was a frequency at least 10× higher than found incontrol DNA templates from FFPE tissues). Urine samples were consideredpositive when the frequency of mutation exceeded 0.1% of alleles (afrequency at least 10× higher than found in control DNA templates fromurine samples of patients without TERT mutations in their primarytumors). All sequencing assays scored as positive were confirmed in atleast one additional, independent PCR and sequence assay.

Statistical Analysis

The data were analyzed using Stata/SE 12 (StataCorp Inc., CollegeStation, Tex.). Pearson's chi-squared test was used for analysis ofassociation of categorical variables. A two-tailed probability <0.05 wasrequired for statistical significance.

Example 2 TERT Promoter Mutation in Papillary and “Flat” NoninvasiveUrothelial Carcinoma

We used a massively parallel sequencing technology to determine thepresence and representation of mutant TERT promoter alleles inurothelial cancers. A graphical depiction of the method is shown in FIG.1 and detailed procedures are provided in the Materials and Methods. Inaddition to revealing whether mutations are present with a population ofDNA templates, this technique provides an accurate determination of thefraction of mutant alleles in the sample. Clinicopathologiccharacteristics of the 76 noninvasive urothelial carcinomas analyzed inthe first phase of this study are summarized in Table 1. They included59 papillary tumors—28 low-grade (pTa LG) and 31 high-grade (pTaHG)—plus 17 “flat” urothelial carcinoma in situ (CIS). These patientswere typical of those with this form of cancer; their average age was 66years and most (82%) were males (Table 1).

TERT promoter mutations were identified in 56/76 (74%) of theseurothelial carcinomas (Table 2). In contrast, none of the eight samplesof adjacent normal urothelium harbored TERT promoter mutations.Additionally, we did not detect TERT promoter mutations in 15 samples ofperipheral blood from the same patients. Twelve of the blood samples andfive of the normal urothelial samples were from patients whose tumorsharbored TERT promoter mutations. These data demonstrate that the TERTpromoter mutations in these patients were unequivocally somatic andlimited to the neoplastic urothelium in the bladder. The predominantalterations were g.1295228C>T (minus strand of chromosome 5, hg19assembly) and g.1295250C>T mutations, which accounted for 75% and 20% ofthe total alterations, respectively. In addition, we identified oneg.1295228C>A mutation and two g.1295242C>T mutations not previouslyreported (Table S1). The mutations were found in all types and grades ofthese early cancers: in 76% of papillary lesions and 65% of flatlesions; in 86% of low-grade and in 68% of high-grade lesions (Table 2).None of these differences among subgroups were statisticallysignificant.

The results described above show that TERT promoter mutations occurearly in bladder cancers and did not correlate with grade or type. Suchearly mutations would not be likely associated with recurrence orprogression, but to evaluate this possibility, our series of samplesincluded cases both with and without recurrence during follow up. InTables 3 and 4, the relationship between TERT promoter mutation statusand tumor recurrence or progression, respectively, are displayed: TERTpromoter mutation status was not associated with likelihood ofrecurrence or progression in any subgroup.

Example 3 TERT Promoter Mutation in Urine Samples

We next evaluated whether TERT promoter mutations could be identified incells in the urine. As noted in the Introduction, urine samples areroutinely taken at follow-up visits following TURB procedures to helpdetermine whether residual tumor cells are present (via cytology orother methods). We first assessed the tumors obtained from 14 patientsundergoing TURB for relatively early (non-muscle invasive) disease. Ofthese, 11 (79%) harbored TERT promoter mutations (Table 5), as expectedfrom the evaluation of the first cohort (Table 2). All of the mutationsin the second cohort were at either g.1295228C>T or g.1295250C>T (Table5).

The 14 patients were monitored for recurrence at subsequent visits.Mutations were assessed in the cell pellets from the urines obtained atthe first follow-up visit after TURB in these 14 patients, as describedin the Materials and Methods. There was a striking correlation betweenthe presence of a TERT promoter mutation in the urine, the presence ofthe mutation in the original tumor, and recurrence. In the three of 14patients without a TERT promoter mutation in their tumor, no mutationwas evident in their urine sample, as expected (Table 5). Of the 11patients in whom a TERT mutation was present in the tumor, sevenpatients were observed to have a mutation in the DNA isolated from theirurine cell pellets; in each case, the mutation was identical to thatobserved in the primary tumor removed via TURB (Table 5). The bladdercancers in each of these seven patients recurred, either at the firstfollow-up or thereafter. The proportion of mutant alleles in the cellspelleted from the urine of these patients was often substantial, rangingfrom 0.17% to 23% with a median of 4.4% (Table 5). We also identified aTERT promoter mutation in a urine sample from which no prior tumor wasavailable; this tumor also recurred (Table 5). In contrast, no TERTmutations were evident in the urine samples of four patients whoseoriginal tumors contained a TERT promoter mutation: the tumors of threeof these patients never recurred while the fourth developed a recurrence3.5 months after the urine sample was collected (Table 5). As shown inTable 6, the presence of detectable TERT promoter mutations in the urinewas strongly associated with recurrence of urothelial carcinoma(P<0.001; Pearson's correlation coefficient=0.87).

Example 4 TERT Promoter Mutation in Fixed Urine Samples

Following routine cytopathology microscopic examination of urine samples(standard of care in primary screen and surveillance for patients withbladder cancern), a residual portion of the urine samples were kept inthe SurePath® preservative. SurePath® Preservative (Becton Dickinson) isan alcohol-based, preservative fluid. The preservation solution servesas a transport, preservative and antibacterial medium for gynecologic(and urine cytology) specimens.

We tested a total of 163 SurePath®-preserved urine specimens. We testedfor TERT promoter mutations in 154 urine SurePath® samples, in additionto those described above in Example 3.

The results were as follows:

TERT Mutation Status in Primary Screen Vs. Surveillance Patients:

Primary screen (hematuria no prior bladder cancer diagnosis)

59 Urine SurePath® samples total

21 TERT promoter mutation positive (36%)

31 TERT promoter mutation negative (53%)

7 Borderline TERT promoter mutation

Surveillance Patients (on follow-up after a diagnosis of bladder cancer)

94 samples total

55 TERT promoter mutation positive (59%)

31 TERT promoter mutation negative (33%)

8 Borderline TERT promoter mutation

As expected, a higher proportion of TERT promoter mutation isencountered in patients with prior diagnosis of bladder cancer(surveillance) compared to those who never had a prior diagnosis ofbladder cancer and who are screened for cancer due to a clinical findinghematuria (primary screen).

TERT Promoter Mutation Status Vs. Routine Cytology Microscopy Diagnosis

All urine cytology samples with positive cytology diagnosis were eitherpositive for TERT promoter mutation (14/17; 82%) or borderline positivefor TERT promoter mutation (3/14; 18%)

Among 54 urine specimens that had atypical cytology diagnosis 55% wereTERT promoter mutation positive; 7% were TERT promoter mutationborderline positive and 38% were negative for TERT promoter mutation

Among 80 urine specimens with a negative cytology diagnosis 40% werepositive for TERT mutation, 9% were borderline for TERT promotermutation, and 51% were negative for TERT promoter mutation.

As one would expect, the rate of TERT positivity was higher in patientswho had a positive diagnosis by cytology, compared to those who hadatypical diagnosis by cytology. The rate of TERT promoter mutationpositivity was in turn higher in patients with a diagnosis of atypicalcytology compared to those that were negative on cytologic exam.

Among all TERT promoter mutation negative samples, none (0%) werediagnosed as positive on cytology. 67% were negative on cytology, 32%were diagnosed as atypical and one was unsatisfactory for examination bycytology.

REFERENCES

The disclosure of each reference cited is expressly incorporated herein.

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We claim:
 1. A method of detecting a somatic mutation and treatingbladder cancer in a human subject comprising: detecting in a nucleicacid obtained from a urine sample of the human subject the presence of asomatic mutation in a promoter of telomerase reverse transcriptase(TERT) gene, wherein the somatic mutation is present at position1,295,228 on the minus strand of chromosome 5 of version hg19 of thehuman genome sequence, wherein the detecting is performed by amplifyinga nucleic acid sequence comprising the TERT promoter, or a portionthereof, to form an amplicon and sequencing the amplicon; and treatingthe human subject with the somatic mutation in the TERT gene for bladdercancer by a therapy chosen from a chemotherapy, a biological agent, asurgery, or a radiation therapy.
 2. The method of claim 1, wherein thehuman subject has a transitional cell carcinoma of the urothelial tract.3. The method of claim 1, wherein the amplicon comprises a position 124bp upstream of the TERT gene transcription start site.
 4. The method ofclaim 1, wherein a C to T mutation is detected in a nucleotide atposition 1,295,228 on the minus strand of chromosome 5 of version hg19of the human genome sequence.
 5. The method of claim 1, wherein thehuman subject has been diagnosed with bladder cancer prior to collectionof the urine sample.
 6. The method of claim 1, wherein a bladder cancerhas been removed from the human subject prior to collection of the urinesample.
 7. A method of detecting, a somatic mutation and treatingbladder cancer in a human subject comprising: detecting in a nucleicacid obtained from a urine sample of a human the presence of a somaticmutation at position 1,295,228 in the promoter of telomerase reversetranscriptase (TERT) gene on the minus strand of chromosome 5 of versionhg19 of the human genome sequence, wherein the detecting is performed byamplifying a nucleic acid sequence comprising the TERT promoter, or aportion thereof, to form an amplicon and sequencing the amplicon; andtreating the human subject with the somatic mutation in the TERT genefor bladder cancer by a therapy chosen from chemotherapy, a biologicalagent, a surgery or a radiation therapy, wherein the human subject hasnot been previously diagnosed with bladder cancer prior to collection ofthe urine sample.
 8. The method of claim 7, wherein the somatic mutationis detected in a low-grade (LG) noninvasive urothelial carcinoma; ahigh-grade (HG) noninvasive urothelial carcinoma or a carcinoma in situ(CIS).
 9. The method of claim 7, wherein the somatic mutation isdetected in a papillary lesion or in a flat lesion.
 10. The method ofclaim 1, wherein the human subject has had surgical excision or othertreatment for the bladder cancer prior to collection of the urinesample.
 11. The method of claim 1, wherein the detecting is used tomonitor the human subject for recurrence of the bladder cancer.
 12. Themethod of claim 1, wherein the human subject has not been previouslydiagnosed with bladder cancer prior to collection of the urine sample.13. The method of claim 1, wherein the therapy is prescribed by amedical practitioner after the somatic mutation is detected.
 14. Themethod of claim 7, wherein the therapy is prescribed by a medicalpractitioner after the somatic mutation is detected.